NO319370B1 - Method of preparing formulation for the treatment or prevention of type 1 diabetes by oral administration of insulin - Google Patents

Abstract

Disclosed herein are methods for treating or preventing a disease in mammals having the characteristics of Type 1 diabetes comprising administering insulin or disease suppressive fragments of insulin or analogs thereof in oral or aerosol dosage forms to said mammals. Also disclosed herein are pharmaceutical formulation or dosage forms for use in the methods.

Description

The present invention relates to a method for producing a formulation for the treatment or prevention of type 1 diabetes by oral administration of insulin. The method according to the invention does not require parenteral administration of therapeutic agents.

Diabetes mellitus is divided into two broad groups based on clinical manifestations, namely the non-insulin-dependent or age-related form, also known as type 2; and the insulin-dependent form that starts in adolescence, also known as type 1. Clinically, the majority of type 2 diabetes that starts in adulthood are obese, with manifestations of clinical symptoms of the disease that usually appear at the age of over 40. In contrast, patients with type 2 diabetes are 1 onset at an early age not overweight for their age and height, with rapid onset of the disease at an early age, often before 30, although type 1 diabetes can occur at any age.

Diabetes mellitus is a metabolic disease state in humans with an incidence of approx. 1% in the general population, a quarter of which are type 1, the insulin-dependent category (Foster, D.W., Harrison's Princi<p>les of Internat Medicine. ch. 114, pp. 661-678, 10th ed., McGraw-Hill, New York). The disease manifests as a series of hormone-induced metabolic abnormalities that can lead to serious, long-term and debilitating complications involving multiple organ systems including the eyes, kidneys, nerves and blood vessels. Pathologically, the disease is characterized by lesions of the basement membranes, which can be demonstrated under the electron microscope.

Type 1 diabetics characteristically show very low or unmeasurable plasma insulin with elevated glucagon. Regardless of the exact etiology, most type 1 patients have circulating antibodies directed against their own pancreatic cells including antibodies against insulin, against the cell cytoplasm of islets of Langerhans and against the enzyme glutamic acid decarboxylase. An immune response specifically directed against beta cells (insulin-producing cells) leads to type 1 diabetes. This specificity is supported by the clinical picture indicated above, since beta cells secrete insulin while alpha cells secrete glucagon.

Current therapeutic treatments for type 1 diabetes include dietary modifications to minimize hypoglycemia that results from the lack of natural insulin, which in turn results from damaged beta cells. Diet is also modified with regard to insulin administration to counteract the hypoglycaemic effects of the hormone. Regardless of the form of treatment, parenteral administration of insulin is required for all type 1 diabetics, hence the term "insulin-dependent" diabetes.

Conventional insulin therapy is limited to the parenteral (i.e. subcutaneous) administration of insulin. Oral administration of insulin has not been possible because the insulin molecule cannot pass through the digestive tract in sufficiently intact form to provide its therapeutic benefit. There has been an ongoing search by those skilled in the art for alternative methods of eliminating or reducing the need for insulin because of the numerous problems associated with subcutaneous administration of the drug. Among the procedures that have been investigated are implantable insulin pumps and transplantation of pancreatic islets.

Because type 1 diabetes usually manifests in adolescence and because subcutaneous delivery of insulin requires strict self-discipline, cooperation is often a serious problem. In addition, the actual act of parenteral administration can be very traumatic for young people. It is difficult for the physician to accurately regulate the amount of insulin required at any given time during the patient's day. Furthermore, it is almost impossible to regulate blood glucose levels in diabetic patients with parenteral insulin to the extent that blood glucose is regulated in normal individuals.

Consequently, in the early stages of treatment of type 1 diabetes, patients often become either hyperglycemic or hypoglycemic because the precise timing of the insulin injections and the required insulin levels are not known. As the treatment progresses, the doctor and, more importantly, the patient adapt to the daily routine, but there is always a risk of ketoacidosis or hypoglycaemia.

In addition, some patients produce antibodies against the injected insulin, although most patients are now treated with human insulin produced by recombinant technology. This can lead to the need for higher insulin doses. Until today, there has been no successful oral dosage form containing insulin.

In the art, there has long been a search for a method to prevent and/or treat type 1 diabetes which does not include parenteral administration of insulin. Various attempts at alternative delivery methods for insulin have been unsuccessful or have not found application at a practical level. E.g. oral insulin, transcutaneous insulin delivery and nasal insulin have not been clinically utilized. Oral insulin does not affect blood glucose levels. Because it has been necessary to inject lipids into mammals receiving nasal insulin to get the insulin through the nasal mucosa, this mode of insulin administration has been limited.

Other dosage forms for the treatment of type 2 diabetes are available (eg oral sulphonylureas). These orally administered agents do not contain insulin, but instead stimulate the pancreas to produce insulin, and are only effective for treating certain forms of type 2 diabetes. In type 1 diabetes, oral agents such as sulfonylureas are ineffective because of the markedly reduced or destroyed beta cell mass (Foster, D.W., "Harrison's Principles of Internal Medicine". ch. 114, p. 668,10 . edition, McGraw-Hill, New York).

Type 1 diabetes is considered a disease of autoimmune etiology (Eisenbarth, G.S., New Engl. J. Med. 314: 1360-1368, 1986). Various animal models are available for the investigation of type 1 diabetes as an autoimmune disease state. These include BB mice (Nakbookda, A.F., et al., Diabetolojac 14: 199-207, 1978) and NOD (non-obese diabetic) mice in which diabetes develops spontaneously (Prochazka et al. Science 237: 286, 1987). Cell-specific, CD4 and CD8 T-lymphocytes have been implicated as causative agents for damaging beta cells, demonstrated by reduced incidence of type 1 diabetes in NOD mice ( J. Exp. Med. 166:823,1987).

Other therapies are being developed for the treatment of autoimmune diseases in general.

Weiner et al., U.S. Patent Application No. 460,852 filed Feb. 21, 1990 (the national step of PCT Application No. PCT/US88/02139, filed June 24, 1988), which is a c-i-p application of US Patent Application No. 065,734 filed June 24, 1987, describes the treatment of autoimmune diseases by oral administration of autoantigens.

Weiner et al., US Patent Application No. 454,486 filed December 20, 1989, describes aerosol administration of autoantigens, disease suppressive fragments of said autoantigens and analogs thereof as an effective method for treating T-cell mediated autoimmune diseases.

Weiner et al., US Patent Application No. 487,732, filed March 2, 1990, describes synergists

(enhancers) for use with oral administration of autoantigens, disease-suppressing fragments and analogues thereof as effective treatments for T-cell mediated autoimmune diseases.

Weiner et al., US Patent Application No. 551,632 filed July 10, 1990, as a c-i-p application under Rule 62 of US Patent Application No. 379,778, filed July 14, 1989 (now assigned), describes methods of preventing or treating uveoretinitis in mammals by oral administration of purified S antigen.

Nagler-Anderson, et. al., ( Proe. Nati. Acad. Sei ( VSA ) 83: 7443-7446,1986), describes the oral administration of collagen to suppress collagen-induced arthritis in a mouse model.

However, none of the above-mentioned treatments for autoimmune diseases can be used in the treatment of type 1 diabetes because the antigens involved in the production and maintenance of type 1 disease have not been identified.

It is therefore an object of the present invention to provide a method for producing a formulation for the treatment or prevention of B-cell destruction in a mammal that has manifested symptoms or is at risk of developing type I diabetes, characterized in that an amount of an agent selected from insulin, autoimmune response suppressive fragments of insulin and analogs thereof, said amount ranging from 1 mg to 100 mg and effective for treating or preventing said β-cell destruction, is mixed with at least one carrier or diluent suitable for administration and then ground, compressed or encapsulation of said formulation into a solid oral formulation, wherein the formulation does not have an endocrinological effect after administration.

Another object of the present invention is to provide a method for producing a formulation suitable for administration by inhalation for the treatment or prevention of B-cell destruction in a mammal that has manifested symptoms or is at risk of developing type I diabetes, in which the drug does not have a endocrinological effect, characterized in that an agent selected from insulin, autoimmune response suppressive fragments of insulin and analogues thereof, is mixed with at least one carrier or diluent and ground into a solid powder comprising particles of an inhalable size range, said particles being packaged in an inhalable solid powder formulation which containing an amount of said agent varying from 1 mg to 1000 mg and effective in treating or preventing said β-cell destruction, wherein said powder formulation does not have an endocrinological effect after its introduction.

A further object of the invention is to provide a method for the preparation of a liquid inhalable dosage form for treating or preventing B-cell destruction in a mammal that has manifested symptoms or is at risk of developing type I diabetes, characterized in that an amount of an agent selected from insulin, autoimmune response suppressive fragments of insulin and analogs thereof are mixed with at least one liquid carrier or diluent to form an aqueous solution, wherein an aliquot of said solution containing an amount of said agent within the range of 1 to 1000 mg is taken which is effective in treating or suppressing said β-cell destruction, and then introducing said solution into a spray or aerosol device and suspending said aliquot of said solution in inhalable aerosol or spray droplets in a carrier gas, wherein said liquid inhalable formulation is without an endocrinological effect after inhalation.

These and other purposes of the present invention will be apparent to the person skilled in the art in light of the following.

It has unexpectedly been discovered that oral administration of insulin is an effective treatment for eliminating or reducing the need for insulin in type 1 diabetics. Oral insulin can prevent or reduce beta cell destruction and thereby reduce or eliminate traditional parenteral insulin therapy.

Orally administrable pharmaceutical preparations containing insulin are prepared and administered to mammals that have manifested symptoms of type 1 diabetes and/or have been diagnosed as having type 1 diabetes. In addition, subjects who are at risk of developing type 1 diabetes (i.e. who have demonstrated a predisposition to develop type 1 diabetes by suitable methods of determination, such as genetic examinations and analysis) are treated with corresponding oral preparations of insulin.

Pharmaceutical preparations for oral or enteral administration to treat type 1 diabetes are prepared from commercially available insulin and a pharmaceutically acceptable carrier suitable for oral administration. The amount of insulin in each dose can be between 1 mg and 1000 mg. However, the total dose required for treatment varies according to the individual. In general, the total amount of insulin required in the practice of the present invention is a far greater dose than the dose administered parenterally to protect an individual afflicted with type 1 diabetes from ketoacidosis.

In addition, an aerosol delivery system can be prepared with equivalent doses of insulin as indicated above with a pharmaceutically suitable carrier or diluent. These and other improvements will be described in the following description, drawings and subsequent claims. Fig. 1 is a graphic representation showing the effects of orally administered porcine insulin on diabetes in NOD mice. Fig. 2 is a graphical representation showing the effects of oral insulin from pigs on serum glucose concentrations after administration of oral insulin in NOD mice. Fig. 3 is a graphical representation showing the effect of oral administration of pancreatic extracts supplied at weekly intervals on the development of diabetes in NOD mice.

All patent applications, patents and literature references referred to herein are incorporated herein by reference in their entirety.

The present invention is aimed at the need for an alternative to existing methods. Because type 1 patients are predominantly young people, it is believed that long-term insulin treatment will no longer be required due to the preservation of unimpaired beta cell function if treatment is initiated at the onset of the disease using the methods of the present invention. If the methods according to the present invention are initiated at a time when a certain beta cell function still exists, parenteral insulin treatment can also be reduced. Accordingly, the present invention provides methods by which long-term insulin therapy can be reduced or no longer required.

It has now surprisingly been discovered that oral and/or aerosol administration of insulin (or disease-suppressing fragments of insulin or analogues thereof) is effective for the treatment and prevention of type 1 diabetes. This is a radical difference from traditional (parenteral) insulin therapy in that insulin is administered to dampen or shut down the host's autoimmune response and not because of its endocrinological (metabolic) effects. Oral administration of insulin has not proven effective in treating any form of diabetes because proteolytic enzymes present in the stomach and digestive system break down the polypeptide before it can reach the blood stream. In addition, intranasal administration of insulin has also not been shown to be effective in the treatment of any form of diabetes.

Without wishing to be bound by any particular theory of action, it is believed that oral or aerosol administration of insulin according to the present invention affects the immunological pathogenesis of type 1 diabetes by eliciting suppressor T cells.

In the following discussions, the following terms shall have the meanings set forth below.

"Treatment" shall mean the treatment of active disease in patients with a certain level of intact insulin-producing beta cells as well as prophylactic administration for use in patients at high risk of developing the disease.

"Oral administration" shall mean both oral administration and enteral administration (direct incubation in the stomach).

"Individuals at risk" for type 1 diabetes shall mean a) individuals who have a blood relative with type 1 diabetes; b) autoantibody-positive individuals without overt type 1 diabetes. These autoantibodies include cytoplasmic islet autoantibodies, insulin antibodies, and glutamic acid decarboxylase autoantibodies; c) individuals with histocompatibility (HLA) type DR3 or DR4DQW8; d) individuals with glucose abnormalities, such as lack of first phase insulin secretion in glucose tolerance tests.

"Mammal" shall mean any organism that has an immune system and is therefore susceptible to type 1 diabetes.

"Active disease" shall mean autoimmune destruction of islet beta cells.

"Aerosol" refers to finely divided solid or liquid particles that can be formed using a pressurized system, such as a nebulizer. The liquid or solid source material contains insulin and/or disease-suppressing fragments of insulin and analogs thereof as defined herein.

"Disease-suppressing fragments" of insulin include any peptide or polypeptide containing partial amino acid sequences or units of insulin and having the ability to treat or prevent a disease having the characteristics of type 1 diabetes. Such fragments need not possess the autoantigenic or endocrinological (metabolic) properties of the entire insulin molecule.

"Analogs" of insulin or disease-suppressing fragments thereof refer to compounds structurally related to insulin or disease-suppressing fragments thereof that have the same biological activity, i.e. the ability to suppress or prevent disease symptoms of type 1 diabetes by oral or aerosol administration. As a non-limiting example, the term includes peptides having amino acid sequences that differ from the amino acid sequence of insulin or disease suppressive fragments thereof by one or more amino acid residues while still preserving the disease suppressive activity of insulin or its ability to prevent or alleviate the symptoms of type 1 diabetes . These analogues do not necessarily have the endocrinological effects of insulin.

According to the present invention, experiments were carried out in which NOD (non-obese diabetic) mice, which develop diabetes spontaneously between 11 and 52 weeks of age, had a lower incidence of diabetes at all doses of orally administered insulin in a dose-dependent manner. In all animals receiving the highest dose of oral insulin (1 mg), 100% of treated animals failed to develop diabetes.

Although it is relatively easy to control the symptoms of type 1 diabetes with parenteral insulin, it is difficult to normalize a patient's blood sugar over 24 hours using traditional insulin therapy given as one or two injections per day. day.

It should be noted that the methods of the present invention will not eliminate the need for parenteral insulin therapy in patients with damaged beta cells who do not produce enough insulin to regulate their blood sugar. However, using the methods of the present invention, newly diagnosed type 1 diabetic patients or those at risk of developing the disease (as defined above), who have substantially intact (undamaged) beta cells, will not go on to develop type 1 diabetes and parenteral insulin administration can be eliminated.

Insulin for use in the method of the present invention can be obtained from numerous commercial sources such as Novo Laboratories (Danbury, CT), Nordic-USA (Rockville, MO) and Eli Lilly and Co. (Indianapolis, IN). Pig-derived insulin, human semi-synthetic insulin (Nordisk-USA) and cloned recombinant insulin (Eli Lilly) can be used when carrying out the method according to the present invention.

Disease-suppressing fragments and analogs of insulin for use in the present invention can be synthesized using well-known solid phase synthesis techniques (Merrifield, R.B. Fed. Proe. Am. Soc. Ex. Biol. 21:412,1962 oe J. Am. Chem. Soc . 85: 2149,1963; Mitchel A.R. et al., J. Am. Chem. Soc. 98: 7357, 1976; Tam, J. et al., J. Am. Chem. Soc. 105: 6442, 1983). Analogues can be constructed by identifying an equivalent amino acid sequence and applying the peptide synthesis techniques described above.

Analogues can be provided by using the known amino acid sequence of insulin as described in "Atlas of Protein Sequence and Structure", Nat. Biochem. Res. Foundation, Volume 5, Pages 209-211.

Disease-suppressing analogs and fragments can also be obtained using recombinant DNA techniques well known in the art.

Disease-suppressing fragments of insulin and analogs thereof can be identified using routine assays using appropriate in vivo systems, such as those set forth in Examples 1-5 below.

Insulin or disease-suppressing fragments or analogues are used orally or enterally, in an amount of between approx. 2 mg per kg body weight of said mammal and approx. 10 mg per kg body weight of said mammal per day, and can be administered in a single-dose form or multiple-dose forms. Preferably, the insulin is administered in an amount between approx. 2.5 mg and approx. 5.0 mg per kg body weight of said mammal per day. The exact amount to be administered will vary depending on the severity and stage of the patient's disease and the physical condition of the patient.

Oral preparations which are used according to the invention may additionally include inert ingredients including pharmaceutically acceptable carriers, diluents, fillers, solubilizing or emulsifying agents and salts as well known in the art. For example can

tablets are formulated according to conventional methods using solid carriers well known in the art. Capsules used can be made from any pharmaceutically acceptable material such as gelatin or cellulose derivatives. Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are also included, such as those described in US Patent No. 4,704,295, issued November 3, 1987, US Patent No. 4,556,552, issued December 3, 1985, US Patent No. 4,309,404, issued January 5, 1982, and US Patent No. 4,309,406, issued January 5, 1982.

Examples of solid carriers include bentonite, silicon oxide and other commonly used carriers. Further non-limiting examples of carriers and diluents which may be used in the compositions of the present invention include saline and any physiologically buffered saline solution such as phosphate buffered saline (PBS) and water.

It should be emphasized that the unit content of active ingredient or ingredients contained in an individual dose of each dosage form does not in itself constitute an effective amount, as the required effective amount can be reached by administering a large number of dosage units.

The preferred method of administration of the dosage forms according to the present invention is oral or enteral. Preferred oral or enteral pharmaceutical preparations or dosage forms can e.g. include between approx. 1 mg and approx. 1000 mg of insulin.

Alternatively, pharmaceutical preparations or dosage forms can also be administered to mammals suffering from diseases having the characteristics of type 1 diabetes in aerosol form. It is expected that lower amounts of insulin, disease-suppressive fragments or analogs thereof will be required when using aerosol administration for the treatment or prevention of type 1 diabetes, as found in the treatment of experimental allergic encephalomyelitis (EAE) with myelin basic protein (MBP) and adjuvant arthritis with collagen as described in US Patent Application No. 454,486 filed December 20, 1989. The amounts of insulin or disease suppressive fragments or analogs thereof that can be administered in an aerosol dosage form will be between approx. 0.1 mg and 10 mg per kg body weight of a mammal per day, and can be administered in a single-dose form or multiple-dose forms. The exact amount to be administered will vary depending on the condition and severity of the patient's illness and the patient's physical condition. Pharmaceutical aerosol formulations may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type well known in the art. Examples of such substances include normal saline solutions, such as physiologically buffered saline solutions, and water.

The method according to the invention can be practiced where insulin or disease-suppressing fragments or analogues thereof are in an aerosol or inhaled form. The insulin and related compounds can be administered as a dry powder or in an aqueous solution. Preferred pharmaceutical aerosol preparations can e.g. include a physiologically acceptable buffered saline solution containing between approx. 1 mg and approx. 1000 mg insulin, disease-suppressing fragments or analogues thereof.

Dry aerosol in the form of finely divided solid particles of insulin, disease-suppressing fragments or analogues thereof which are not dissolved or suspended in a liquid are also useful in the practice of the present invention. The insulin can be in the form of dust powders and include finely divided particles that have an average particle size of between approx. 1 and 5 µm, preferably between 2 and 3 µm. Finely divided particles can be prepared by pulverization and sieve filtration using techniques well known in the art. The particles can be administered by inhaling a predetermined amount of the finely divided material, which can be in the form of a powder.

Specific, non-limiting, examples of the carriers and/or diluents useful in the pharmaceutical aerosol preparations include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline solutions, pH 7.0 - 8.0.

The pharmaceutical preparations can be administered in the form of an aerosol spray using e.g. an atomizer as described in US Patent No. 4,624,251 issued November 25, 1986; 3,703,173 issued November 21, 1972; 3,561,444 issued February 9, 1971 and 4,635,627 issued January 13, 1971. The aerosol material is inhaled by the object being treated.

Other aerosol delivery systems, such as the pressurized metered dose inhaler (MDI) and the dry powder inhaler described in Newman, S.P. "Aerosols and the Lung", Clarke, S.W. and Davia, D. eds. pages 197-224, Butterworths, London, England, 1984, can be used in carrying out the present invention.

Aerosol delivery systems of the type described herein are available from numerous commercial sources including Fisons Corporation (Bedford, MA), Schering Corp. (Kenilworth, NJ) and American Pharmoseal Co., (Valencia, CA).

It is expected that the methods according to the present invention are particularly suitable for use in pediatric or young patients who develop type 1 diabetes or in the first year after the onset of hyperglycaemia, where the secondary effects of the disease (such as vascular damage, kidney damage and diabetic retinopathy) are not is totally manifested. These are also the patients where the traumatic effects of daily insulin injections are usually most noticeable.

As shown in the examples set forth below, oral administration of insulin to NOD mice reduced the number of these mice that subsequently went on to develop diabetes at all doses tested. At the highest dose used (1 mg), none of the mice developed diabetes. In addition, as shown in Example 2, the effects of oral insulin were not caused by a reduction in serum glucose concentrations in these animals and, therefore, were not a metabolic effect. As shown in Example 3 below, in addition, feeding NOD mice with pancreatic extract also led to a reduction in the number of animals that developed diabetes, albeit to a lesser extent than for those receiving oral insulin.

Initial experiments have shown that NOD mice given 1 mg of insulin had fewer inflammatory immune system cells surrounding the pancreas (a condition known as insulinitis) than PBS-fed NOD mice (data not shown). Therefore, oral administration of insulin to NOD mice appears to affect the infiltration of immune system cells into the pancreas and consequently halt disease progression by preserving beta cell function.

The present invention is described further below in working examples which are intended to illustrate the present invention without reducing its scope.

Example 1:

The effect of administration of porcine insulin on spontaneous diabetes in NOD mice

NOD (non-obese diabetic) mice were obtained from Taconic Laboratories (Germantown, MA) at 4 weeks of age. There were a total of 30 animals in each of the groups mentioned below at the beginning of the experiment. At 414 weeks of age, animals were orally administered porcine insulin (Novo Laboratories, Danbury, CT). Each animal received two treatments orally twice a week until 9 weeks of age. The animals were then given either saline solution (control) or 10 micrograms, 100 micrograms or 1 milligram of porcine insulin. The animals were gavaged with an 18 gauge ball tip needle (Popper and Sons, Inc., New Hyde Park, NY). Starting at 10 weeks, the animals were then fed once a week for a total of 33 weeks. Beginning at 12 weeks, the urine was examined weekly for the presence of glucose using "Glucosuria" test strips (Eli Lilly, Indianapolis, IN). If an animal had +3 or higher on the glucose tolerance test, a serum sample was taken and blood glucose was measured using a glucose analyzer (Beckman). If blood glucose was 220 or higher, the animals were classified as diabetic and then euthanized.

As shown in Fig. 1, no diabetes developed in any of the animals given 1 mg insulin, while diabetes developed in the control group and in the 10 microgram and 100 microgram groups in a dose-dependent manner. The p-value comparing the 1 mg supply with the control is p<0.008, while in the 100 microgram supply is p<0.09.

The normal blood insulin level in an animal is between 120 to 170 mg/decaliter or mg/dl. The blood sugars in the animals fed PBS that developed diabetes were: 575,485,459, 500, 375,400, 362, 395,480 mg/dl.

The blood sugar in the animals given 10 micrograms of insulin that developed diabetes was: 330, 420, 315, 459, 520, 487 mg/dl.

The blood sugar in animals given 100 micrograms of insulin that became diabetic was:

290, 600, 450, 500 mg/dl.

The animals that received 1 mg of insulin were also examined for blood sugar. None of the animals developed diabetes as measured by urine testing, but blood sugar was examined as a confirmation. Blood sugar in 10 representative animals from the group supplied with 1 mg insulin was: 174, 128, 125, 125, 145, 123, 136, 155, 115, 130 mg/dl.

Example 2:

Glucose after oral insulin in fasting 7-week-old NOD mice

To determine whether any endocrinological (i.e., direct lowering of blood glucose) effects occurred after administration of insulin, 7-week-old NOD mice (5 mice per group) were administered 1 mg of porcine insulin or 1 mg of myelin basic protein (obtained from Pel Freez, Rogers, AR) as a control protein. Blood glucose was then measured over the next 24 hour period. As a positive control, a separate group of animals was subcutaneously injected with 20 micrograms of porcine insulin. All animals were kept in a fasted state.

As shown in Fig. 3, the animals given subcutaneous insulin had an immediate drop in glucose levels with a reduction of approx. 80 mg% which persisted over a 4 hour period and then slowly returned to normal. Animals receiving oral insulin or oral myelin basic protein had no drop in blood glucose; on the other hand, there was an increase in blood glucose which is probably associated with the protein load. Over the next 24 hours, there was a gradual decrease in blood glucose in all groups of animals since the animals were in a fasted state. This was observed both in animals given insulin and animals given myelin basal protein.

These results demonstrate that there were no endocrinological (metabolic) effects from the administration of oral insulin. Based on published data regarding the generation of immunological tolerance and immunological effects after oral administration of proteins, these results strongly suggest that oral administration of insulin affects the immunological pathogenesis of diabetes in the NOD mice.

Example 3:

Effect of oral administration of pancreatic extract administered at weekly intervals

An identical experiment to Example 1 was performed in which NOD mice (14-17 per group) were administered a homogenized pancreas derived from Lewis rats in 0.5 ml of PBS. As shown in Fig. 3, there was a reduction in the development of diabetes in extract-fed animals compared to controls. Nevertheless, the effect is not as dramatic and complete as that observed in animals given insulin. These results demonstrate some effect of pancreatic tissue delivery and highlight the potency of the insulin trials described above.

Example 4:

Effect of aerosol administration of insulin on diabetes in NOD mice

Insulin (0 to 1 mg as in Examples 1 and 2) is administered to NOD mice as described above in Examples 1 and 2 except in aerosol form using a nebulizer.

It is expected that insulin in aerosol form will be more effective in preventing the manifestation of symptoms (i.e. hypoglycemia) of type 1 diabetes in the treated mice.

Example 5:

Treatment of initial stages of diabetes in NOD mice

Insulin (0 to 1 mg as in Examples 1 and 2) is administered to NOD mice suffering from hypoglycemia in an oral and aerosol form. Treatments begin at 12-20 weeks of age, by which time serum glucose levels are at hypoglycemic concentrations in a certain percentage of animals before the onset of diabetes.

It is expected that both oral and aerosol administration of insulin will lead to a reduction in serum glucose concentrations to close to normal levels and preservation of beta cell function.

Claims (10)

1. Method for the preparation of a formulation for the treatment or prevention of JJ cell destruction in a mammal having manifested symptoms or at risk of developing type I diabetes, characterized in that an amount of an agent selected from insulin, autoimmune response suppressive fragments of insulin and analogs thereof , said amount varies from 1 mg to 100 mg and is effective for the treatment or prevention of said β-cell destruction, is mixed with at least one carrier or diluent suitable for introduction and then grinding, compressing or encapsulating said formulation into a solid oral formulation, wherein the formulation does not have an endocrinological effect after introduction. 2. Method according to claim 1, characterized in that said solid formulation is selected from a tablet, a capsule and a caplet. 3. Method according to claim 1, characterized in that the carrier or diluent is selected from gelatin, cellulose derivatives, bentonite and silica. 4. Process for the preparation of a formulation suitable for administration by inhalation for the treatment or prevention of 6-cell destruction in a mammal that has manifested symptoms or is at risk of developing type I diabetes, in which the drug does not have an endocrinological effect, characterized in that an agent selected from insulin, autoimmune response suppressive fragments of insulin and analogs thereof, are mixed with at least one carrier or diluent and ground into a solid powder comprising particles of an inhalable size range, said particles being packaged in an inhalable solid powder formulation containing an amount of said agent varying from 1 mg to 1000 mg and effective in treating or preventing said β-cell destruction, wherein said powder formulation does not have an endocrinological effect after its introduction. 5. Method according to claim 4, characterized in that it further includes driving said solid powder formulation into an inhalable solid powder suspension in a carrier gas using an aerosol device. 6. Method according to claim 4, characterized in that the solid powder formulation is an aerosol. 7. Method according to claim 4, characterized in that the solid powder formulation is further characterized in that the particles have an average particle size of 1 to 5 microns. 8. Method according to claim 4, characterized in that the solid powder formulation is further characterized in that the particles have a preferred average particle size of 2 to 3 microns. 9. Method according to claim 6, characterized in that the aerosol contains an amount of said agent which corresponds to 0.1 mg to 10 mg per kg body weight of the mammal. 10. Method for the preparation of a liquid inhalable dosage form for treating or preventing B-cell destruction in a mammal having manifested symptoms or at risk of developing type I diabetes, characterized in that an amount of an agent selected from insulin, autoimmune response suppressive fragments of insulin and analogs thereof are mixed with at least one liquid carrier or diluent to form an aqueous solution, wherein an aliquot of said solution containing an amount of said agent within the range of 1 to 1000 mg effective to treat or suppress said P - cell destruction, and then introducing said solution into a spray or aerosol device and suspending said aliquot of said solution in inhalable aerosol or spray droplets in a carrier gas, wherein said liquid inhalable formulation is without an endocrinological effect after inhalation.